The Ataxia telangiectasia Gene Product Is Required for Oxidative Stress-induced G 1 and G 2 Checkpoint Function in Human Fibroblasts

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond to t-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G(1) and G(2) phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage

Site-selective nuclear magnetic relaxation time in a superconducting vortex state

The temperature and field dependences of the site-selective nuclear spin relaxation time T_1 around vortices are studied comparatively both for s-wave and d-wave superconductors, based on the microscopic Bogoliubov-de Gennes theory. Reflecting low energy electronic excitations associated with the vortex core, the site selective temperature dependences deviate from those of the zero-field case, and T_1 becomes faster with approaching the vortex core. In the core region, T_1^{-1} has a new peak below the superconducting transition temperature T_c. The field dependence of the overall T_1(T) behaviors for s-wave and d-wave superconductors is investigated and analyzed in terms of the local density of states. The NMR study by the resonance field dependence may be a new method to probe the spatial resolved vortex core structure in various conventional and unconventional superconductors.Comment: 14Pages, 26 figures, revte

Quantitative detection of 4-hydroxyequilenin–DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody

Estrogen–DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN–DNA adducts. Although the formation of 4-OHEN–DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN–DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN–dA adducts and of 4-OHEN–dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose–response between known amounts of 4-OHEN–DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/108 bases in 1 µg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN–DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN–DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN–DNA adducts in mammalian cells

An Obscure Case of Hepatic Subcapsular Hematoma

Spontaneous liver bleeding is often reported in preeclampsia. It is otherwise rare and has been linked to gross anatomical lesions and coagulopathy. We report a case of subcapsular hematoma of the liver without any apparent lesion and in the absence of coagulopathy. A 41-year-old male, paraplegic for 16 years, presented to the emergency department 3 days after sudden onset of right upper quadrant and shoulder pain. He had been on vitamins and 5,000 units subcutaneous heparin 12-hourly at the nursing home for the last month. He was in no distress, afebrile, with stable vitals. Physical examination showed a diverting colostomy, tender hepatomegaly and sacral decubiti. A fecal occult blood test was negative. There was spastic paraplegia below the level of T12. Two days after admission, the patient was afebrile and hemodynamically stable. PTT, PT, liver profile, BUN and creatinine were all normal, however his hemoglobin had dropped from 11.3 to 7.6 g/dl. An abdominal CT scan revealed an isolated 9.0 × 1.8 cm subcapsular hematoma. The patient received blood transfusion in the intensive care unit and was discharged 7 days later. In conclusion, spontaneous liver hemorrhage occurs in the nonobstetrical population in the setting of gross anatomical lesions or coagulopathy. This is the first report of an isolated subcapsular liver hematoma

Effects of gap anisotropy upon the electronic structure around a superconducting vortex

An isolated single vortex is considered within the framework of the quasiclassical theory. The local density of states around a vortex is calculated in a clean type II superconductor with an anisotropy. The anisotropy of a superconducting energy gap is crucial for bound states around a vortex. A characteristic structure of the local density of states, observed in the layered hexagonal superconductor 2H-NbSe2 by scanning tunneling microscopy (STM), is well reproduced if one assumes an anisotropic s-wave gap in the hexagonal plane. The local density of states (or the bound states) around the vortex is interpreted in terms of quasiparticle trajectories to facilitate an understanding of the rich electronic structure observed in STM experiments. It is pointed out that further fine structures and extra peaks in the local density of states should be observed by STM.Comment: 11 pages, REVTeX; 20 PostScript figures; An Animated GIFS file for the star-shaped vortex bound states is available at http://mp.okayama-u.ac.jp/~hayashi/vortex.htm

Human MLH1 Protein Participates in Genomic Damage Checkpoint Signaling in Response to DNA Interstrand Crosslinks, while MSH2 Functions in DNA Repair

DNA interstrand crosslinks (ICLs) are among the most toxic types of damage to a cell. For this reason, many ICL-inducing agents are effective therapeutic agents. For example, cisplatin and nitrogen mustards are used for treating cancer and psoralen plus UVA (PUVA) is useful for treating psoriasis. However, repair mechanisms for ICLs in the human genome are not clearly defined. Previously, we have shown that MSH2, the common subunit of the human MutSα and MutSβ mismatch recognition complexes, plays a role in the error-free repair of psoralen ICLs. We hypothesized that MLH1, the common subunit of human MutL complexes, is also involved in the cellular response to psoralen ICLs. Surprisingly, we instead found that MLH1-deficient human cells are more resistant to psoralen ICLs, in contrast to the sensitivity to these lesions displayed by MSH2-deficient cells. Apoptosis was not as efficiently induced by psoralen ICLs in MLH1-deficient cells as in MLH1-proficient cells as determined by caspase-3/7 activity and binding of annexin V. Strikingly, CHK2 phosphorylation was undetectable in MLH1-deficient cells, and phosphorylation of CHK1 was reduced after PUVA treatment, indicating that MLH1 is involved in signaling psoralen ICL-induced checkpoint activation. Psoralen ICLs can result in mutations near the crosslinked sites; however, MLH1 function was not required for the mutagenic repair of these lesions, and so its signaling function appears to have a role in maintaining genomic stability following exposure to ICL-induced DNA damage. Distinguishing the genetic status of MMR-deficient tumors as MSH2-deficient or MLH1-deficient is thus potentially important in predicting the efficacy of treatment with psoralen and perhaps with other ICL-inducing agents

The Genome of a Pathogenic Rhodococcus: Cooptive Virulence Underpinned by Key Gene Acquisitions

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi